1. Field of the Invention
The invention relates to a process for preparing dipeptides and more particularly, to a process for the preparation of dipeptides represented by the following formula (I) which comprises reacting N-blocked aspartic acid represented by the following formula (II) with a lower alkyl ester of phenylalanine represented by the following formula (III) in the presence of an organic solvent mixture and immobilized enzyme, ##STR1## wherein X is a protective group used in peptide synthesis and Y is a lower alkyl group.
2. Description of the Prior Art
Generally, it is well known that protease can be used for forming peptide bonds in the reverse reaction of protein decomposition. A number of conventional methods have been proposed for favoring the reverse reaction toward synthesis of dipeptides. U.S. Pat. No. 4,165,311 to Isowa et al discloses a process for producing an addition compound which includes reacting a dipeptide and amino acid ester in an aqueous medium in the presence of a protease. However, only a soluble enzyme may be used. Generally, such a soluble enzyme is not only expensive but is unstable in soluble form so that it can only be utilized in very limited industrial applications. In order to overcome such limitations, research has been conducted for several years. As a result, it is possible to improve the enzyme stability by immobilizing techniques. However, in such an addition compound forming system, it is practically impossible to use any kind of immobilized enzymes due to the difficulties of enzyme separation therefrom. U.S. Pat. No. 4,284,721 to Oyama et al. discloses that a water-containing immobilized metallo-proteinase in an organic solvent immiscible with water is used to favor the peptide bond formation. J. Org. Chem., 46, p. 5241 (1981) published by Oyama et al discloses that, due to an immediate transfer of the product toward the water-immiscible organic solvent, the reaction rate as well as the equilibrium yield are relatively good although they are lower than those in the aqueous medium.
Furthermore, although the Oyama et al reference employs an immobilized enzyme which is stable in the organic solvent, since the enzyme requires a certain water content, the water-immiscible organic solvent inevitably requires to contain two phases. However, such a two phase medium limits the continuous operation of synthetic processes, particularly, a process of the packed bed column reactor and stirred tank reactor. In the stirred tank reactor, an attrition of the enzyme necessarily occurs. Furthermore, even though the enzyme is in an immobilized form, it is not satisfactorily stable.
To avoid these disadvantages of the water-immiscible solvent medium, the use of an organic solvent miscible with water has been considered. However, in such a medium, a micro-environmental change between the water disposed around the immobilized enzymes and the organic solvent causes feedback inhibition so that the reaction rate inevitably decreases.
On the other hand, however, the fact that an organic cosolvent can shift the reversible peptide forming reaction toward synthesis has been reported by Homandberg et al (Biochem. 17 (24), 5220 (1978)).